Genetically based diseases are life threatening and they require lifelong treatment. There are currently over 30,000 single base pair mutations like cystic fibrosis, Down syndrome (in human beings), hemophilia, sickle cell anemia etc. many of which offer insufficient treatments, sometimes no treatment option is available . Gene therapy is one of the advanced therapeutic methods aiming essentially to cure the disease at gene level and to reverse the disease phenotype. Previously researchers have used viral vectors, but have not been very successful because the viral vectors lack specificity with regard to integration sites and there was a high incidence of integration near proto-ontogenetic sites, which result in leukemia in majority of patients. Viral vectors also can trigger immunogenic response that can be fatal. On top of these serious risks, random integration of a full-length gene could result in abnormal gene expression, if the promoter is not endogenous.
Gene editing or targeted nucleotide exchange arose as an alternative strategy that corrects the underlying disease causing mutation in the endogenous gene, so that gene expression pattern remains unchanged. Gene editing became popular in the world of research as an alternative method to treat genetic disorders and to get rid of life-long drug treatments . Currently, Targeted nucleotide exchange (TNE) reaction utilizes a short single strand of DNA, an oligonucleotide (ODN) that pairs with the targeted gene . Previously scientists have used mammalian cells, HCT116 (Human colon tumor cell line), as model cell line in gene editing. These cell lines were cultured and harvested at different conditions, ODNs were introduced into the cells through electroporation, and the results were analyzed using a combination of cell cycle and flow cytometry analyses to know the efficiency of correction or treatment. While studying the mechanism of TNE, recent studies have developed an interest in the function of MutS homolog2 (MSH2), a mismatch repair protein  and p53, a genome surveillance protein. MSH2 has been shown to interfere with the recombination between the sequences in TNE reaction through anti-recombinase activity , while P53 has known to cause suppression of homologous recombination (HR).
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Statement of the problem: As genetically based diseases are life threatening and require lifelong treatment, it is a novel idea to cure those diseases at the gene level itself by correcting altered genes. There are several obstacles like DNA damaging proteins (P53 AND MSH2) which hinder this process. We can apply certain procedures to get rid of those obstacles one such method is using RNAi to knock down DNA damage protein.
Gene editing is the repair of single base mutation in mammalian genes that are directed by short single-stranded DNA oligonucleotides (ssODN). Previously researchers have used HCT116-19 (clone19) cell line that has non-sense mutation at position + 67 resulting in nonfunctional eGFP (enhanced green fluorescent protein). To correct the single base changes in chromosomal DNA, gene editing is employed by using a mutated eGFP ssODNs with sequence of 5'-GAAGCACTGCACGCCGGAGGTCA GGGT-3'; and targeting sequence is 5'GAAGCACTGCACGCCATATGTCAGGGT-3' . Several configurations of synthetic oligonucleotides have been used in gene editing including short and long unmodified DNA molecules, chimerical 2'O-Me-RNA-DNA hairpin oligonucleotides . The editing oligonucleotides are specially designed to restore the fluorescence of the eGFP. In such system, editing is easily evaluated, by visual inspection where corrected cells are identified as green cells under fluorescent microscope and, quantitatively by flow cytometry analyses .
Currently gene editing utilizes a short strand of DNA oligonucleotide (ssODN) that pairs with the target gene. Most commonly used ODN in mammalian system is approximately 72 nucleotide in length with a single and centrally located mismatch. It has a modification with three terminal phosphor thioate linkages on both ends of ODN in place of phophodiester back bone, to protect from nuclease degradation . ssODNs of this fashion have been known to increase the correction efficiencies of 1-2%. ODN-induced gene editing is not necessitated in viral vector delivery systems. ODN delivery in to cells is performed either with the aid of cationic liposomes that coat DNA or with electroporation, which essentially permeabilizes the membrane allowing ODN to enter in to cells.
Mechanism of ODN-induced gene editing: Research utilizing many cell model systems with different reporter genes has suggested four possible mechanisms. There is evidence that all the four mechanisms support each other, but they may not be mutually exclusive. Initial step in gene editing reaction is; ODN pairing to its target site which is heavily influenced by homologous recombination protein, Rad51 [6, 7]. Each of the four mechanisms initiates ODN pairing to its target DNA causing subsequent D-loop formation . The mechanisms differ based on how the D-loop, a reaction intermediate, is resolved to produce a corrected gene. In direct repair mechanism, [Fig1] D-loop serves as cellular beacon of DNA that can accelerate repair proteins to resolve the mismatch. The non-complimentary pairing resolved by ODN directs the conversion of mutant endogenous base. When ODN leaves the target site, cell is left with a mismatch between newly corrected DNA base and endogenous mutant complimentary base. Another DNA repair step resolves this mismatch by correcting the complimentary base to wild type gene status .
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Another possible mechanism in gene editing reaction involves ODN pairing to the target site while the gene being transcribed actively. As RNA polymerase complex occupies the transcribed strand, non-transcribed strand remains unbound providing an opportunity for ODN to bind its complimentary site. The RNA polymerase can switch from transcribed strand to ODN and then back to transcribed strand, creating a corrected mRNA without altering DNA sequences. This would lead to functional protein production, creating a phenotypic change, which will be observed without genotypic change. This mechanism can also explain the strand bias, where in most cases there is higher correction efficiencies in case of ODN compliments to non-transcribed strand than to transcribed strand . ODN may be directly incorporated in to target gene [Fig1.1]. The direct integration model depend on the formation of D-loop and subsequent replacement of D-loop with ODN.
Researchers have made some manipulations to obtain higher correction efficiencies in gene editing. Such manipulations of cell cycle have determined that progression of S-phase is extremely important for higher correction efficiencies. Targeting cells in mid S-phase leads to elevated corrections, whereas targeting cells in early or in late S-phase have diminished correction efficiencies. Progression of S-phase can be achieved by adding either aphidicoline or dodeoxy cytidine (ddc) to cells, 24hours prior to targeting with ODN.
Mismatch repair pathway (MMR) was involved in this DNA repair event, which is inhibitory to the ODN-induced gene editing reaction in mammalian cells . MSH2, a mismatch repair protein and p53, a genome surveillance protein inhibits gene editing and ultimately leads to reduced correction efficiency. In DNA repair pathway, MSH2 and p53 combines with ODN and inhibits ODN pairing to target DNA.
Fig1: Pathways of gene editing
Also known as tumor protein 53 or protein 53 is a tumor suppressor gene that is encoded by TP53gene [22, 23]. P53 is important in multi cellular organisms where it regulates cell cycle and thus functions as tumor suppressor, which involved in preventing cancer. Referring to role in conserving stability by preventing genome mutation, p53 is described as guardian of the genome. The name p53 relates to its molecular mass as it runs a 53-kilodalton (kDa) protein on SDS-PAGE .
The official name of MSH2 is 'mutS homolog 2'a mismatch repair protein. MSH2 provides an instruction for making a protein that plays an important role in DNA repair. This protein fixes mistakes that were made when DNA replicated in preparation for cell division. MSH2 protein combines with another two proteins MSH6 and MSH3 forming an active protein complex that identifies places on DNA where mistakes have been made during DNA replication.
Cell sorting: Single green fluorescent gene edited cells were sorted on a FACS (Fluorescent Activated Cell Sorting) analyses. Events recorded based on Forward scatter/side scatter, live/dead.
Small interfering RNA (siRNA) is one of the latest additions to the repertoire of specific gene silencing agents. These molecules silence the specific gene expression. RNA interference or RNA silencing occurs in wide variety of eukaryotic organisms. It is triggered by dsRNA precursors, which vary in length and origin . Short inhibited RNAs of 21-28 nucleotide length were formed from dsRNA. According to their origin or function, three types of naturally occurring small RNAs have been described, interfering RNA (RNAi), micro RNA (miRNA), repeat associated short inhibited RNA (rasiRNA). By using RNA-templated RNA, polymerization or hybridization of overlapping transcripts dsRNA can be produced. Such dsRNA give rise to siRNA, which guide the mRNA degradation. Finally, artificial introduction of dsRNA or siRNA is adopted as a tool for suppression of specific proteins expression. RNA silencing mechanism is first discovered as an antiviral mechanism to protect organisms against RNA viruses.
Structure of siRNA: siRNA molecules are short double stranded RNA duplexes with symmetric 2-3 nucleotide 3'overhangs the 5' phosphate and 3'hydroxylgroups (Fig 2.1).
Structure of siRNA
Fig 2.1: siRNA with 3' hydroxyl and 5'phosphate groups
The siRNA pathway
RISC (RNA inducing silencing complex)
Degradation of Mrna
Normal functions of p53:
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p53 has many anti cancer mechanisms and plays an important role in apoptosis, genetic stability and inhibition of angiogenesis. p53 works through many mechanisms in its anti-cancer role. It activates DNA repair proteins when DNA has damage. It can induce growth arrest by holding the cell cycle at G1/S phase, if p53 holds the cell cycle at this stage DNA damage proteins will have much time and the cell will be allowed to continue cell cycle. If DNA damage is irreparable, p53 can initiate apoptosis (Fig: 3.2).
Activated p53 binds with DNA and activates the expression of several genes encoding for p21. p21 is important in cell cycle transition as a stop signal for cell division. A tumor p53 will no longer bind to DNA, there will be no p21 production and uncontrollable cell division leads to tumor formation. If TP53 gene is damaged tumor suppression function will be lost. Loss of p53 can result in genomic instability that creates aneuploidy phenotype .
Effects of p53 on gene editing: Apart from tumor suppression and cell cycle checkpoints activation, p53 is also involved in homologous recombination (HR) in gene editing and inhibits the HR proteins like Rad51, which is important for homologous recombination . Furthermore, p53 is recruited to stalled replications to prevent uncontrolled homologous exchanges . P53 acts as antagonist and prevents the initiation of oligonucleotide-directed gene repair, reducing the level of eGFP positive cells and ultimately leads to reduction in correction efficiency.
P53 expression knockdown: In gene editing p53 expression can be temporarily knocked down by using siRNA. SiRNA can be introduced in to cells prior to protein extraction; cell extracts are prepared, proteins are separated on a 10%SDS-PAGE gel and electro blotted to a nitrocellulose membrane. The membrane incubated with p53, a mouse monoclonal antibody against total p53, and secondary is goat anti-mouse horseradish peroxidase (HRP) conjugated antibody followed by chemilumniscent detection. Actin expression can be used as an internal control to normalize protein levels and can be used using beta actin primary followed by anti-goat HRP. Suppression of p53 can be observed .
Effects of MSH2 on gene editing: MSH2 plays a suppressive role in gene editing by precluding the oligonucleotide (ODN) annealing to the target gene. Furthermore, MSH2 has an anti-recombinase activity during homologous recombination (HR) and interact with several proteins involved in the regulation of cell cycle . MSH2 inhibits the recombination between sequences during HR. MSH2 combines with the ODN and prevents its pairing to target DNA.
Knockdown of MSH2 by siRNA: Transient knock down of MSH2 is very important to increase correction efficiency in gene editing. Permanent knockdown of MSH2 will prevent its normal function and predispose cells to cancer. siRNA having specific sequence against MSH2 mRNA can be used to down regulate MSH2. Wild-type cells (containing eGFP mutant) transfected with siRNA, allow siRNA to act for 24 hours and knockdown the expression of MSH2. Protein samples from these cells and subjected to western blot to check reduced MSH2 levels. MSH2 suppressed cells can be used in gene editing and targeted with ODN. Correction efficiency can be analyzed through flow cytometry and flourecent activated cell sorting.
Null hypothesis (H0): RNAi does not have any effect on DNA damaging proteins in Gene Editing and ultimately it is not going to affect correction efficiency.
Alternative hypothesis (HA): RNAi can have effect on DNA damaging proteins in gene editing and it is going to increase correction efficiency by knock downing DNA damage proteins.
Materials and Methods:
Cell line and culture conditions:
HCT116 Cell lines were obtained from ATCC (American Type Cell culture).The integrated HCT116 clone 19(HCT116-19) is produced through the integration of peGFP-n3 vector that has a mutated eGFP gene. This mutated eGFP gene has a non sense mutation at +67 position resulting in non-functional eGFP protein. HCT 116-19 cells are cultured in McCoy's5AModified medium that supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% pencillin/streptomycin. Cells are maintained at 370c and 5% co2.
Oligonucleotide (ODN) design:
The targeting ODNs are designed as 72-mers that complemented either with the non-transcribed (NT) or transcribed (T) strand of the target mutant eGFP gene. Each ODN has three phosphorothioate linkages on either end of it to prevent nuclease degradation. The ODNs that are designed to elicit correction of the gene (72NT and 72T) have a central mismatch which would direct conversion of the mutant stop codon to the wild-type eGFP tyrosine, thereby allowing expression of functional eGFP.
As a first step cells were synchronized with 2 μM aphidicolin for 24 h in regular growth medium. On day 2 Cells were trypsinized and harvested by centrifugation. Cells were resuspended to get a final concentration of 2.5 - 107 cells/mL in serum-free media and 100 μL (1*106 cells) transferred to a 4 mm gap cuvette. ODN was added to a final concentration of 4 μM and cells were electroporated (electroporation conditions: 250 V, 13 ms, 2 pulses, 1 s interval) using BTX Electro Square Porator. The electroporated cells then transferred to a 6 well plate and allowed to recover in regular growth medium for 24 h at 37 °C.
Flow cytometry and immunofluorescence:
eGFP fluorescence was measured by a flow cytometer 24 h after electroporation. Cells were harvested by trypsinization and resuspended in FACS buffer (0.5% BSA, 2 mM EDTA, 2 μg/mL propidium iodide in PBS). The correction of a single base mutation in an integrated copy of the eGFP gene results in the emergence of green fluorescence in HCT116-19 cells. 106 cells are electroporated in the presence of the ODN after 24 h of pre-treatment with 2 μM aphidicolin; the cells are processed for FACS before doing this we can check for emergence of green fluorescence which indicates the presence of cells containing a wild-type (corrected) eGFP gene. Corrected cells are quantitated through FACS by counting live fluorescent cells and dividing by the total number of live cells. This method generate a value referred to as the correction efficiency (CE), which is often represented as a percentage (%) Correction efficiency.
Targeted cells are allowed to recover for 24 h. Cells were then harvested by trypsinization and 104 cells were isolated and pelleted. The pellet dissolved in PBS and washed, finally suspended in 20 μL of 1x PBS. Cells and agarose were combined in a 1:10 ratio and immediately poured onto a COMET slide (avoid solidification of agar). This mixture was allowed to cool for 10 min at 4 °C in the dark. After 10 minutes slide should be immersed in lysis solution at 4 °C for 60 min. In the next step slides were immersed in a freshly prepared alkaline solution, pH 11.0, for 60 min at the room temperature and covered with a black lid to avoid light. Slides were rinsed in TBE buffer and transferred to an electrophoresis apparatus. TBE was added to cover slides and the electrophoresis unit was run at 1 V/cm for 15 min. After 15 minutes Slides were submerged in 70% ethanol for 5 min and allowed to dry in the dark overnight. On the next day a few drops of SYBR Green I was added to each sample and the slides were covered with a cover slip and sealed with nail polish on four sides. Slides were examined on a Zeiss LSM 510. Number and percent of COMETs were determined for each field and take the mean for each treatment.
RNAi knockdown procedure: HCT116-19 cells were seeded at a density of 2.5-106 cells/100 mm dish and electroporated with oligonucleotide and 2uM P 53RNAi in a total volume of 100 μl. To determine the level of P53 protein after RNAi treatment, after 8 and 12 hours, the cells were collected and total protein was extracted in 2X sample buffer at 95°C and incubated at 95°C with intermittent mixing at full speed for a total of 12 minutes. The samples were then centrifuged for 10 minutes at 13,200 rpm at 4°C and the supernatant was collected. The total protein concentration was measured with the BCA assay using BSA as a standard. Protein concentrations were determined, separated on a 10% SDS-PAGE gel according to standard procedures, and proteins were transferred to a PVDF membrane .The blot was blocked for 2 hours at RT in 5% nonfat milk with 0.5% Tween-20 and then incubated with a primary antibody against P53 or a goat polyclonal antibody against β-actin. The appropriate species specific HRP-conjugate antibody was used as a secondary antibody to observe the proteins.
Limitations and Delimitation: Though we will get some higher correction efficiencies by knock dowing the P53 protein significant amount of DNA damage is always expected in this method. Furthermore while introducing ODN through electroporation rapid cell death was there, so still need to find more sophisticated method of introducing ODN in to cells
Significance: In this study if once we come up with significant correction efficiencies it means mutated version turned to wild type , without any medical treatment we can able to treat the genetic disorders at the gene level itself which saves lot of money that spending on medical treatment and lot of time.